Qtrap 4500 system

To measure the endogenous concentrations of JA and SA, root samples of about 100–200 mg were homogenised twice with 80% (V/V) cold methanol and shaken overnight in 4°C darkness. Dissolution, filtration, storage and quantification of combined extracts are described by Sun et al. (2014) (link). SA and JA content were measured by HPLC-MS/MS performed by AB SCIEX QTRAP 4500 system (AB SCIEX, Foster City, CA, United States) as described by Zhou et al. (2018) (link). The experiment was repeated 3 times with more than 3 seedlings in the sample.

Quantitative analysis was performed on Shimadzu RRLC instrument coupled with a QTRAP 4500 system (AB SCIEX, Redwood, CA, USA); which was equipped with a binary high-pressure solvent delivery system (LC-30AD pump, Shimadzu Corporation, Kyoto, Japan). The separation was carried out on a Thermo Scientific Hypersil GOLD C18 column (100 mm × 2.1 mm, 1.9 μm) with 40 °C. Mobile phase was composed of 0.1% formic acid in water (A) and acetonitrile (B) with a fast gradient program as follows: 5% to 40% (B) in 0 to 3 min, 40% (B) in 3 to 5 min, 40% to 80% (B) in 5 to 5.5 min, 80% (B) in 5.5 to 7 min, 80% to 5% (B) in 7 to 7.1 min, 5% (B) in 7.1 to 9 min. The flow rate was set at 0.3 mL/min and the injection volume was 10 uL.
All analytes were confirmed and quantified by tandem mass spectrometry operating in electrospray positive ionization mode (ESI+) with MRM mode. The MS parameters were optimized and set as follows: Ion spray voltage at 5500 V, the turbo spray temperature at 500 °C, curtain gas (CUR) at 35 psi, nebulizer gas (GS1) at 50 psi, heater gas (GS2) at 50 psi, collision gas at 6 psi, and dwell time at 20 ms. The optimized declustering potential (DP) and proper collision energy (CE) are listed in Table 2.
Data acquisition and procession were performed on Analyst 1.6 software (AB SCIEX, Redwood, CA, USA).

The collected blood was centrifuged at 4000 rpm at 4 °C for 10 min to collect plasma samples, which were stored at below −70 °C until analysis. All frozen plasma samples were thawed at 20 °C prior to analysis. The concentration of TS in the plasma was analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry (Agilent 1290 Infinity II, Agilent Technologies, Santa Clara, CA, USA; QTRAP^® 4500 System, AB SCIEX, Framingham, MA, USA). A 2-µL sample was injected into the C18 column (4.6 × 50 mm, 1.8 μm). The mobile phase consisted of acetonitrile and 0.1% formic acid, provided as isocratic elution at a flow rate of 0.3 mL/min. The total run time was 3 min.

MGO-conjugated adducts of chrysin were purified by using a recycle HPLC with a gradient system (0–25%, (MeOH)) as the eluent to obtain CS-mono-MGO adduct (5.14 mg) and CS-di-MGO adduct (4.83 mg). Additionally, isolated MGO-conjugated adducts of chrysin were identified as follows: (1) Liquid chromatography mass spectrometry (LC-MS/MS): The LC eluent was introduced into the ESI interface. The positive ion polarity mode was utilized for the ESI ion source. LC-MS/MS spectrum obtained using a QTRAP 4500 system (AB SCIEX, Darmstadt, Germany) with curtain gas 35 psi, ion spray voltage 5500 volts, source temperature 650 °C, nebulizer gas 55 psi, heater gas 55 psi, and scan range of 100–500 Da; (2) Nuclear magnetic resonance (NMR): Approximately 3.0–5.0 mg of each compound was dissolved in 600 μL of dimethyl sulfoxide (DMSO)-d6 and distributed in 3-mm NMR tubes. ¹H and ¹³C-NMR spectra and correlation NMR spectra were obtained using an Avance DPX 400 spectrometer (Bruker, Billerica, MA, USA). Spectra were obtained at operating frequencies of 400 (¹H) and 100 MHz (¹³C) with DMSO-d6, and tetramethylsilane was used as an internal standard.